Isolated Hepatocytes (isolated + hepatocyte)

Distribution by Scientific Domains


Selected Abstracts


Efficient hepatocyte engraftment and long-term transgene expression after reversible portal embolization in nonhuman primates,

HEPATOLOGY, Issue 3 2009
Ibrahim Dagher
The feasibility of ex vivo gene therapy as an alternative to liver transplantation for the treatment of liver metabolic diseases needs to be analyzed in large animal models. This approach requires appropriate gene transfer vectors and effective hepatocyte engraftment. Lentiviral vectors have the ability to transduce nondividing differentiated cells, such as hepatocytes, and portal vein occlusion increases hepatocyte engraftment. We investigated whether reversible portal vein embolization combined with ex vivo lentivirus-mediated gene transfer is an effective approach for successful hepatocyte engraftment in nonhuman primates and whether the transgene remains expressed in the long term in transplanted hepatocytes in situ. Simian hepatocytes were isolated after left lobe resection, and the left and right anterior portal branches of animals were embolized with absorbable material. Isolated hepatocytes were labeled with Hoechst dye or transduced in suspension with lentiviruses expressing green fluorescent protein under the control of the human apolipoprotein A-II promoter and transplanted via the inferior mesenteric vein. The whole procedure was well tolerated. The embolized liver was revascularized within 2 weeks. The volume of nonembolized liver increased from 38.7% ± 0.8% before embolization to 55.9% ± 1% after embolization and hepatocytes significantly proliferated (10.5% ± 0.4% on day 3 after embolization). Liver repopulation after transplantation with Hoechst-labeled hepatocytes was 7.4% ± 1.2%. Liver repopulation was 2.1% ± 0.2% with transduced hepatocytes, a proportion similar to that obtained with Hoechst-labeled cells, given that the mean transduction efficacy of simian hepatocyte population was 34%. Transgene expression persisted at 16 weeks after transplantation. Conclusion: We have developed a new approach to improve hepatocyte engraftment and to express a transgene in the long term in nonhuman primates. This strategy could be suitable for clinical applications. (HEPATOLOGY 2009.) [source]


Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°C

LIVER INTERNATIONAL, Issue 3 2003
Alan J. Wigg
Abstract: Isolated hepatocytes in suspension provide a number of advantages for use in bioartificial liver device, however, poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. We therefore investigated the integrity and differentiated function of isolated rat hepatocytes under conditions of mild hypothermia. Isolated hepatocytes were suspended in a bicarbonate buffered saline medium, supplemented with glucose and bovine serum albumin (BSA), and maintained for 48 h at 25 °C on a rotary shaker under an atmosphere of 95% O2 and 5% CO2. Under these conditions there was no significant decline in cell viability and good preservation of cellular morphology on transmission electron microscopy for at least 24 h. Isolated hepatocytes in suspension at 25 °C were also able to maintain normal Na + and K + ion gradients. The cellular energy status ([ATP], ATP/ADP ratio, cytoplasmic and mitochondrial redox potentials), metabolic function (urea synthesis and ammonia removal), albumin synthesis and phase I and phase II drug detoxification activity of these cells were also maintained for at least 24 h post isolation. These observations demonstrate the robust nature of mildly hypothermic isolated hepatocytes in suspension and encourage further studies re-examining the feasibility of using this cell preparation in bioartificial livers. [source]


Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes

FEBS JOURNAL, Issue 15 2002
Ulrike Krause
Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming biosynthetic pathways. [source]


Cytosolic calcium regulates liver regeneration in the rat,

HEPATOLOGY, Issue 2 2010
Laura Lagoudakis
Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES,expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES,expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells. Conclusion: Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle. (HEPATOLOGY 2010;) [source]


Mrp2 modulates the activity of chloride channels in isolated hepatocytes

HEPATOLOGY, Issue 1 2002
Xinhua Li
Adenosine triphosphate binding cassette family transport proteins are important organic ion transporters in hepatocytes but these molecules may also exhibit other functions. In the present study we have measured the effects of substrates of the canalicular organic ion transporter multidrug resistance associated protein 2 (Mrp2) on chloride channel activation and cell volume regulation. We found that substrates such as leukotriene D4, 17-,-estradiol glucuronide, and the leukotriene inhibitor MK-571 accelerated the activation of chloride channels by cell swelling and activated chloride channels in cytokine-pretreated hepatocytes. Two conjugated estrogens that are not Mrp2 substrates did not produce this effect. Hepatocytes derived from a strain of transport-deficient rats (TR,), which lack Mrp2 expression, showed none of these substrate effects. Coincident with their ability to activate channels, the Mrp2 substrates increased the rate of volume regulatory decrease by approximately 50% (P < .01), confirming that enhanced channel activation under this condition stimulated volume regulation. In TR-hepatocytes the Mrp2 substrate had no effect on volume regulation. In conclusion, Mrp2 plays a role in regulation of chloride channel function by reducing the lag time necessary for channel activation and consequently accelerating the process of cell volume regulation. Substrates of Mrp2 affect the ability of the protein to interact with chloride channels. These findings represent an alternative function of Mrp2 in hepatocytes. [source]


Regulation of CYP1A2 by histone deacetylase inhibitors in mouse hepatocytes

JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2004
Bohwan Jin
Abstract Cytochrome P450 1A2 (CYP1A2) is constitutively expressed in the mouse liver, but the constitutive expression progressively declines to an undetectable level in isolated hepatocytes. In this study, CYP1A2 was induced in hepatocytes exposed to the histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), but only well after constitutive CYP1A2 expression was silenced. However, cotreatment with the arylhydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) and either TSA or SB reduced the induction of CYP1A2 with the same time course as TSA or SB increased its induction. These results suggest that histone modification is involved in CYP1A2 regulation in hepatocytes through pathways that are independent of AhR. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:131,132, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20017 [source]


Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherin

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008
Masato Nagaoka
Abstract Rearrangement of cell,cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E-cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E-cadherin-rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E-cadherin-immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E-cadherin-mediated cell adhesion vary with the state of differentiation. J. Cell. Biochem. 103: 296,310, 2008. © 2007 Wiley-Liss, Inc. [source]


Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cells

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2001
Toru Mizuguchi
Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood-forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow-derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co-cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell,cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcirption factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3-dioxygenase, are improved in hepatocyte/BMSC co-cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro. © 2001 Wiley-Liss, Inc. [source]


P-selectin mediates leukocyte rolling in concanavalin-A-induced hepatitis

LIVER INTERNATIONAL, Issue 5 2005
Sandra March
Abstract: Concanavalin- A (Con-A)-induced hepatitis is an experimental model of human autoimmune hepatitis characterized by leukocyte activation and infiltration of the liver. The aim of the present study was to evaluate the role of P-selectin on leukocyte,endothelial interactions within the hepatic microvasculature in response to Con-A. Methods: The study was performed in P-selectin-deficient mice and wild-type mice pretreated with anti-P-selectin blocking monoclonal antibody (mAb) or vehicle. After 2 h of Con-A (20 mg/kg i.v.) or PBS administration, leukocyte rolling and adhesion and the index of sinusoidal perfusion were evaluated using the intravital microscopy technique in the liver. Apoptosis was determined by flow cytometry analysis of caspase-3 activity assayed on freshly isolated hepatocytes. Results: Con-A induced a significant increase in leukocyte rolling, mainly located at the central venule (2.1±0.4 vs 0.6±0.2 cells/min in wild-type mice treated with vehicle) and less marked, but still significant, in portal venules. This was associated with a significant increase in leukocyte adhesion. In P-selectin-deficient mice treated with Con-A, leukocyte rolling in portal and central venules was markedly reduced. However, leukocyte adhesion was only partially attenuated. A few sinusoids were perfused in wild-type mice treated with Con-A (26%). The percentage of perfused sinusoids was significantly higher in P-selectin-deficient mice (45%; P<0.05 vs wild-type). Similar effects were noted after the simultaneous injection of Con-A and anti-P-selecting mAb in wild-type mice. After Con-A treatment, apoptosis was markedly reduced in isolated hepatocytes of P-selectin-deficent mice (37±7% vs 75±5% in wild type). Conclusion: The results of this intravital microscopy study clearly demonstrate that P-selectin is involved in the initial leukocyte rolling that leads to the development of Con-A-induced liver injury. [source]


Characteristics of murine histidinaemia and its potential for genetic manipulation

LIVER INTERNATIONAL, Issue 4 2004
N. Mellor
Abstract: Background: Histidinaemia is an autosomal recessive disorder affecting the hepatic enzyme histidine ammonia lyase (histidase) resulting in elevated plasma and urinary histidine and is prototypic of a series of hepatic cytosolic enzyme defects. Aims: To characterise the physiology of murine histidinaemia with respect to histidine excretion and catabolism, and explore the potential for manipulating cellular and whole body histidase metabolism by gene transfer. Materials and Methods: We studied his/his mice which have a G to A substitution in the gene encoding histidase, using both in vitro transduction of isolated hepatocytes by lipofection with wild-type histidase cDNA, and in vivo transduction of whole liver using a retroviral construct. Results and Conclusion: Histidase cDNA expression restored histidase activity in vivo and in vitro towards normal levels, demonstrated both at the cellular level and by whole body metabolic studies, establishing the potential of this model for the development of new gene therapeutic approaches. [source]


Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°C

LIVER INTERNATIONAL, Issue 3 2003
Alan J. Wigg
Abstract: Isolated hepatocytes in suspension provide a number of advantages for use in bioartificial liver device, however, poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. We therefore investigated the integrity and differentiated function of isolated rat hepatocytes under conditions of mild hypothermia. Isolated hepatocytes were suspended in a bicarbonate buffered saline medium, supplemented with glucose and bovine serum albumin (BSA), and maintained for 48 h at 25 °C on a rotary shaker under an atmosphere of 95% O2 and 5% CO2. Under these conditions there was no significant decline in cell viability and good preservation of cellular morphology on transmission electron microscopy for at least 24 h. Isolated hepatocytes in suspension at 25 °C were also able to maintain normal Na + and K + ion gradients. The cellular energy status ([ATP], ATP/ADP ratio, cytoplasmic and mitochondrial redox potentials), metabolic function (urea synthesis and ammonia removal), albumin synthesis and phase I and phase II drug detoxification activity of these cells were also maintained for at least 24 h post isolation. These observations demonstrate the robust nature of mildly hypothermic isolated hepatocytes in suspension and encourage further studies re-examining the feasibility of using this cell preparation in bioartificial livers. [source]


Influence of dietary palm oil on growth, tissue fatty acid compositions, and fatty acid metabolism in liver and intestine in rainbow trout (Oncorhynchus mykiss)

AQUACULTURE NUTRITION, Issue 4 2005
J. FONSECA-MADRIGAL
Abstract This study aimed to investigate the effects of dietary crude palm oil (CPO) on fatty acid metabolism in liver and intestine of rainbow trout. Triplicate groups of rainbow trout for 10 weeks at 13 °C were fed on diets in which CPO replaced fish oil (FO) in a graded manner (0,100%). At the end of the trial, fatty acid compositions of flesh, liver and pyloric caeca were determined and highly unsaturated fatty acid (HUFA) synthesis and fatty acid oxidation were estimated in isolated hepatocytes and caecal enterocytes using [1- 14C]18:3n-3 as substrate. Growth performance and feed efficiency were unaffected by dietary CPO. Fatty acid compositions of selected tissues reflected the dietary fatty acid composition with increasing CPO resulting in increased proportions of 18:1n-9 and 18:2n-6 and decreased proportions of n-3HUFA, 20:5n-3 and 22:6n-3. Palmitic acid, 16:0, was also increased in flesh and pyloric caeca, but not in liver. The capacity of HUFA synthesis from 18:3n-3 increased by up to threefold in both hepatocytes and enterocytes in response to graded increases in dietary CPO. In contrast, oxidation of 18:3n-3 was unaffected by dietary CPO in hepatocytes and reduced by high levels of dietary CPO in enterocytes. The results of this study suggest that CPO can be used at least to partially replace FO in diets for rainbow trout in terms of permitting similar growth and feed conversion, and having no major detrimental effects on lipid and fatty acid metabolism, although flesh fatty acid compositions are significantly affected at an inclusion level above 50%, with n-3HUFA reduced by up to 40%. [source]


Equally potent inhibitors of cholesterol synthesis in human hepatocytes have distinguishable effects on different cytochrome P450 enzymes

BIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2000
L.H. Cohen
Abstract Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC50 values: 0.2,8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug,drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6,-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S -mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1,-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O -dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 µM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC50 values around 200 µM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC50 value: 4 µM). Using the assay of testosterone 6,-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC50 values of about 200 µM and a little more by C and A (IC50 around 100 µM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug,drug interaction is considered. Copyright © 2000 John Wiley & Sons, Ltd. [source]